Philip M. Mullineaux

Photosynthetic electron transport regulates the expression of cytosolic ascorbate peroxidase genes in Arabidopsis during excess light stress.

Exposure of Arabidopsis plants that were maintained under low light (200 mumol of photons m-2 sec-1) to excess light (2000 mumol of photons m-2 sec-1) for 1 hr caused reversible photoinhibition of photosynthesis. Measurements of photosynthetic parameters and the use of electron transport inhibitors indicated that a novel signal transduction pathway was initiated at plastoquinone and regulated, at least in part, by the redox status of the plastoquinone pool. This signal, which preceded the photooxidative burst of hydrogen peroxide (H2O2) associated with photoinhibition of photosynthesis, resulted in a rapid increase (within 15 min) in mRNA levels of two cytosolic ascorbate peroxidase genes (APX1 and APX2). Treatment of leaves with exogenous reduced glutathione abolished this signal, suggesting that glutathione or the redox status of the glutathione pool has a regulatory impact on this signaling pathway. During recovery from photooxidative stress, transcripts for cytosolic glutathione reductase (GOR2) increased, emphasizing the role of glutathione in this stress.

Technical Focus:A guide to Agrobacterium binary Ti vectors

We are grateful to the following for agreeing to deal with requests for plasmids and strains: Elizabeth Hood, Jonathan Jones, Bob Ludwig, Madan Bhattacharya, Pal Maliga, Csaba Koncz, Dirk Inze, Eugene Nester, Gynheung An, Mike Bevan and Alex McCormac. R.P.H. and P.M.M. gratefully acknowledge the support of the Biotechnology and Biological Sciences Research Council for support in the development of pGreen. We wish to thank Rod Casey for critical reading of the manuscript.

Evidence for a Direct Link between Glutathione Biosynthesis and Stress Defense Gene Expression in Arabidopsis

The mutant regulator of APX2 1-1 (rax1-1) was identified in Arabidopsis thaliana that constitutively expressed normally photooxidative stress-inducible ASCORBATE PEROXIDASE2 (APX2) and had ≥50% lowered foliar glutathione levels. Mapping revealed that rax1-1 is an allele of γ-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), which encodes chloroplastic γ-glutamylcysteine synthetase, the controlling step of glutathione biosynthesis. By comparison of rax1-1 with the GSH1 mutant cadmium hypersensitive 2, the expression of 32 stress-responsive genes was shown to be responsive to changed glutathione metabolism. Under photo-oxidative stress conditions, the expression of a wider set of defense-related genes was altered in the mutants. In wild-type plants, glutathione metabolism may play a key role in determining the degree of expression of defense genes controlled by several signaling pathways both before and during stress. This control may reflect the physiological state of the plant at the time of the onset of an environmental challenge and suggests that changes in glutathione metabolism may be one means of integrating the function of several signaling pathways.

Elevated Glutathione Biosynthetic Capacity in the Chloroplasts of Transgenic Tobacco Plants Paradoxically Causes Increased Oxidative Stress

Glutathione (GSH), a major antioxidant in most aerobic organisms, is perceived to be particularly important in plant chloroplasts because it helps to protect the photosynthetic apparatus from oxidative damage. In transgenic tobacco plants overexpressing a chloroplast-targeted γ-glutamylcysteine synthetase (γ-ECS), foliar levels of GSH were raised threefold. Paradoxically, increased GSH biosynthetic capacity in the chloroplast resulted in greatly enhanced oxidative stress, which was manifested as light intensity–dependent chlorosis or necrosis. This phenotype was associated with foliar pools of both GSH and γ-glutamylcysteine (the immediate precursor to GSH) being in a more oxidized state. Further manipulations of both the content and redox state of the foliar thiol pools were achieved using hybrid transgenic plants with enhanced glutathione synthetase or glutathione reductase activity in addition to elevated levels of γ-ECS. Given the results of these experiments, we suggest that γ-ECS–transformed plants suffered continuous oxidative damage caused by a failure of the redox-sensing process in the chloroplast.

Improving water use in crop production.

Globally, agriculture accounts for 80–90% of all freshwater used by humans, and most of that is in crop production. In many areas, this water use is unsustainable; water supplies are also under pressure from other users and are being affected by climate change. Much effort is being made to reduce water use by crops and produce ‘more crop per drop’. This paper examines water use by crops, taking particularly a physiological viewpoint, examining the underlying relationships between carbon uptake, growth and water loss. Key examples of recent progress in both assessing and improving crop water productivity are described. It is clear that improvements in both agronomic and physiological understanding have led to recent increases in water productivity in some crops. We believe that there is substantial potential for further improvements owing to the progress in understanding the physiological responses of plants to water supply, and there is considerable promise within the latest molecular genetic approaches, if linked to the appropriate environmental physiology. We conclude that the interactions between plant and environment require a team approach looking across the disciplines from genes to plants to crops in their particular environments to deliver improved water productivity and contribute to sustainability.

Lesion simulating disease 1 is required for acclimation to conditions that promote excess excitation energy

The lsd1 mutant of Arabidopsis fails to limit the boundaries of hypersensitive cell death response during avirulent pathogen infection and initiates unchecked lesions in long day photoperiod giving rise to the runaway cell death (rcd) phenotype. We link here the initiation and propagation of rcd to the activity of photosystem II, stomatal conductance and ultimately to photorespiratory H2O2. A cross of lsd1 with the chlorophyll a/b binding harvesting-organelle specific (designated cao) mutant, which has a reduced photosystem II antenna, led to reduced lesion formation in the lsd1/cao double mutant. This lsd1 mutant also had reduced stomatal conductance and catalase activity in short-day permissive conditions and induced H2O2 accumulation followed by rcd when stomatal gas exchange was further impeded. All of these traits depended on the defense regulators EDS1 and PAD4. Furthermore, nonphotorespiratory conditions retarded propagation of lesions in lsd1. These data suggest that lsd1 failed to acclimate to light conditions that promote excess excitation energy (EEE) and that LSD1 function was required for optimal catalase activity. Through this regulation LSD1 can influence the effectiveness of photorespiration in dissipating EEE and consequently may be a key determinant of acclimatory processes. Salicylic acid, which induces stomatal closure, inhibits catalase activity and triggers the rcd phenotype in lsd1, also impaired acclimation of wild-type plants to conditions that promote EEE. We propose that the roles of LSD1 in light acclimation and in restricting pathogen-induced cell death are functionally linked.

Signal transduction in response to excess light: getting out of the chloroplast

Plants are continually in danger of absorbing more light energy than they can use productively for their metabolism. Acclimation to environmental conditions therefore includes the development of mechanisms for dissipating or avoiding the accumulation of such excess excitation energy. Acclimation could be controlled by many signal transduction pathways that would be initiated by the perception of excess excitation energy both inside and outside the chloroplast. Recent studies in related areas provide models of how these signalling pathways could operate in acclimation to excess light. Components of photosynthetic electron transport chains, reactive oxygen species, redox-responsive protein kinases, thiol-regulated enzymes, chlorophyll precursors and chloroplast-envelope electron transport chains all have roles in these models.

Subcellular distribution of multiple forms of glutathione reductase in leaves of pea (Pisum sativum L.)

On sodium-dodecyl-sulfate polyacrylamide gels, purified glutathione reductase (GR; EC 1.6.4.2) from the leaves of two- to three-week-old pea (Pisum sativum L. cv. Birte) seedlings was represented by a single band with an apparent molecular weight of 55 kilodaltons. This polypeptide was resolved to multiple isoforms by two-dimensional electrophoresis. Fractionation of protoplasts and purification of subcellular organelles has shown that enzyme activity is associated with the chloroplasts, mitochondria and cytosol (in this order, approx. 77%, 3%, and 20% of the total activity). Distinct multiple isoforms of the enzyme, which differed in isoelectric point and were compartment-specific, were resolved from purified mitochondria and chloroplasts. The latency of the glutathione reductase activity which co-purified on Percoll gradients with the mitochondrial marker enzyme, cytochrome-c oxidase (EC 1.9.3.1.), indicated that this enzyme was within the mitochondrion. The mitochondrial glutathione reductase activity was strongly dependent on NADPH and not NADH.

Chloroplast Signaling and LESION SIMULATING DISEASE1 Regulate Crosstalk between Light Acclimation and Immunity in Arabidopsis

Plants are simultaneously exposed to abiotic and biotic hazards. Here, we show that local and systemic acclimation in Arabidopsis thaliana leaves in response to excess excitation energy (EEE) is associated with cell death and is regulated by specific redox changes of the plastoquinone (PQ) pool. These redox changes cause a rapid decrease of stomatal conductance, global induction of ASCORBATE PEROXIDASE2 and PATHOGEN RESISTANCE1, and increased production of reactive oxygen species (ROS) and ethylene that signals through ETHYLENE INSENSITIVE2 (EIN2). We provide evidence that multiple hormonal/ROS signaling pathways regulate the plants response to EEE and that EEE stimulates systemic acquired resistance and basal defenses to virulent biotrophic bacteria. In the Arabidopsis LESION SIMULATING DISEASE1 (lsd1) null mutant that is deregulated for EEE acclimation responses, propagation of EEE-induced programmed cell death depends on the plant defense regulators ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4). We find that EDS1 and PAD4 operate upstream of ethylene and ROS production in the EEE response. The data suggest that the balanced activities of LSD1, EDS1, PAD4, and EIN2 regulate signaling of programmed cell death, light acclimation, and holistic defense responses that are initiated, at least in part, by redox changes of the PQ pool.

Light perception in plant disease defence signalling

Light is a predominant factor in the control of plant growth, development and stress responses. Many biotic stress responses in plants are therefore specifically adjusted by the prevailing light conditions. The plant cell is equipped with sophisticated light-sensing mechanisms that are localised inside and outside of the chloroplast and the nucleus. Recent progress has provided models of how the signalling pathways that are involved in light perception and in defence could operate and interact to form a plant defence network. Such a signalling network includes systems to sense light and regulate gene expression. Photo-produced H(2)O(2) and other reactive oxygen species in the cell also play an essential role in this regulatory network, controlling biotic and abiotic stress responses.